Helicobacter pylori (Hp) is the leading cause of duodenal ulcers and gastric cancer worldwide. Unfortunately, existing antibiotics no longer effectively eradicate Hp infection and cure these ailments. The development of new treatments will be greatly aided by insights into the pathogenesis of Hp. Virulence of Hp appears to be directly linked to the pathogen's ability to glycosylate proteins. Although Hp's glycans and glycosylation machinery are linked to pathogenesis, Hp's glycome remains poorly understood. The long-term goal of this project is to develop chemical tools that enable fundamental studies of bacterial glycosylation, particularly with respect to human disease. The objective of this application is to expand the recently introduced technique of metabolic oligosaccharide engineering (MOE) to study and perturb glycoproteins of the pathogen Hp. MOE is a powerful chemical method that installs detectable reporters into glycans, ultimately permitting their visualization, enrichment, identification, and perturbation. The central hypothesis of the application is that MOE will reveal new Hp glycoproteins, facilitate the identification of glycosylation machinery, and serve as a platform for targeting unique Hp glycans. The rationale for the proposed research is twofold: once Hp's glycoproteins and glycosylation machinery have been characterized, new targets of therapeutic intervention will be revealed. Further, once Hp is targeted based on its unique glycans, the stage will be set for pre-clinical trials to evaluate this strategy as a means to treat Hp infection. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Characterize Hp's glycoproteins and glycosylation machinery;and 2) Chemically target Hp based on its unique glycans. Under the first aim, an already proven MOE approach will be utilized to enrich Hp's glycoproteins, and then the enriched proteins and the glycans that modify them will be identified by mass spectrometry analysis. Further, Hp deletion mutants lacking putative glycosyltransferases will be constructed, and their glycoprotein profile will be assessed using MOE to identify the genes responsible for glycoprotein synthesis. Under the second aim, the cell surface sugar pseudaminic acid, which is essential for Hp's pathogenesis yet absent from humans, will be targeted to selectively inactivate Hp. Azide-labeled pseudaminic acid residues on Hp will be targeted with therapeutic phosphines, and then damage to Hp will be assessed. This approach is innovative, because it capitalizes on applying a well-established technique to a highly novel system. The proposed research is significant, because it is expected to initiate the development of effective glycosylation-based therapeutic strategies to eradicate Hp infections. Moreover, the chemical approach developed here will be broadly applicable in other bacteria and greatly facilitate the study of bacterial glycoproteins. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under-investigated area of bacterial biology that has potential applicability to understanding the pathogenesis of Helicobacter pylori infection, as well as providing new targets for therapeutic interventions that will aid patients suffering from ulcers and gastric cancer. The proposed research has relevance to public health, because the molecules to be investigated are tied to pathogenesis. Thus, the findings are ultimately expected to be applicable to eradicating bacterial infection and improving human health.